© 2000 Wiley-Liss, Inc. Cytometry 40:88–101 (2000) Original Articles Polarization of Scatter and Fluorescence Signals in Flow Cytometry Charles L. Asbury, Jeanne L. Uy, and Ger van den Engh* Department of Molecular Biotechnology, University of Washington, Seattle, Washington Received 1 November 1999; Revision Received 31 January 2000; Accepted 16 February 2000 Background: The pulses of light scatter and fluorescence Results: Measurements of cells stained with a variety of measured in flow cytometers exhibit varying degrees of dyes illustrate that fluorescence polarization occurs fre- polarization. Flow cytometers are heterogeneously sensi- quently in flow cytometry. tive to this polarization, depending on the light source(s), Conclusions: Consequences for quantitative cytometry the optical layout, and the types of mirrors and filters are discussed, and the use of the “magic angle” to make a used. Therefore, fluorescence polarization can affect ap- flow cytometer insensitive to fluorescence polarization is parent intensity ratios between particles and interfere proposed. Cytometry 40:88–101, 2000. with schemes for interlaboratory standardization. © 2000 Wiley-Liss, Inc. Methods: We investigate the degree to which polariza- tion affects common flow cytometry measurements. Our technique for determining polarization differs from previ- ous methods because complete distributions of intensity Key terms: fluorescence polarization; anisotropy; transi- versus polarization angle are measured, rather than inten- tion dipole; fluorescein (FDA, FITC); phycoerythrin (PE); sities at just two orthogonal polarization angles. Theoret- thiazole orange (TO); ethidium bromide (EB); propidium ical models for scatter and fluorescence are presented and iodide (PI); TOTO-1; ethidium homodimer (ETHD-2); verified by making polarization measurements of calibra- YOYO-1; quantitative flow cytometry (QFCM) tion beads. Light has three qualities: color, intensity, and polariza- tation and polarization sensitivity of the detector. Ideally, tion. In cytometry, we are usually concerned with the first one would present the complete angular distributions of two properties only. We select color with a suitable filter intensity and polarization emitted by an object of interest. and measure the intensity of light that passes the filter. Such detailed specifications of experimental conditions The polarization of the light is usually ignored, despite the and results are not commonly practiced by the flow cy- fact that the intensity measurement may depend on the tometry community. degree and direction of polarization. Most cytometers are A cavalier treatment of polarization would be justified (unintentionally) polarization sensitive. Therefore, the ap- if anisotropy played no role in flow measurements. parent intensity of a light source can be changed by rotating the detection optics around the collection axis. Of even greater consequence is the fact that polarization Clarification of terms: degree of polarization: quantitative measure of the amount of polarization, P value; direction of polarization: the direc- of a fluorescent source is an indication of anisotropy: the tion of the electric field of a photon; polarization state: refers to both the object emits different amounts of light in different direc- degree and direction of polarization; emission anisotropy: total emission tions. Two identical detectors looking at the same object is different depending on the direction of observation; anisotropy in the distribution of dipoles: distribution of dipoles is not spherically symmet- from different vantage points may give different light ric. intensity readings. With anisotropic light emission and Grant sponsor: NSF Science and Technology Center; Grant number: BIR polarization-sensitive detectors, the question, “How bright 9214821; Grant sponsor: NIH; Grant number: T32 GM00035-05. is the particle?” has an ambiguous answer. Avoiding this *Correspondence to: Ger van den Engh, Department of Molecular Biotechnology, University of Washington, Box 357730, Seattle, WA ambiguity requires specifying the polarization state of the 98195-7730. incident light, the direction of observation, and the orien- E-mail: [email protected] POLARIZATION IN FLOW CYTOMETRY 89 However, there are good reasons to suspect that flow THEORY cytometry is affected by anisotropy. Flow cytometry Different mechanisms are responsible for the anisot- almost exclusively employs coherent, highly polarized ropy of scatter and fluorescence. Although geometrically light sources, so the scatter and fluorescence pulses complex, the scattering of polarized light by small trans- measured in flow experiments are very likely to be parent particles is described well by the rules of classical anisotropic. In the few cases where investigators have optics: reflection, refraction, diffraction, and interference. looked for fluorescence polarization, it has been ob- In contrast, the light-matter interactions that lead to fluo- served. Early measurements of polarization in a flow rescence must be analyzed at the molecular level. These cytometer were made by Arndt-Jovin et al. (1,2). They considerations lead to different predictions for the aniso- originally used the method to observe changes in the tropy of fluorescence and scatter signals that can be tested rotational freedom of membrane-bound dyes (i.e., mem- by experiment. brane fluidity). Later (3), they used the method to verify The following section describes the physical processes the occurrence of energy transfer between labeled cell- that cause light scattering and fluorescence emission using surface receptors. Other investigators have used similar simple models. It will be shown that, under conditions methods to measure the fluorescence polarization of typical in flow cytometry, both scatter and fluorescence intracellular fluorescein (4–7) and of various DNA- and are anisotropic, exhibiting significant variations in inten- membrane-bound dyes (8–15), because polarization sity with both the angle of observation and the polariza- provides information on the cellular context of these tion sensitivity of the detector. The models are then used molecules. Measurements of fluorescence polarization to make quantitative predictions about the direction and of peripheral blood cells have been reported to be degree of polarization expected in flow experiments. useful for the study and diagnosis of cancer (16) and Alzheimer’s disease (17). Besides these experiments, Angular Distribution of Scattered Light which deliberately aimed to induce and utilize signal polarization, there are few observations of polarization When a laser beam hits a small particle, incident light is under “normal” conditions. The degree to which anisot- scattered unequally in many directions. The intensity of the scatter at a given angle of observation depends mainly ropy occurs in ordinary flow experiments is not docu- on the properties of the particle: its refractive index, its mented. shape, and especially its size. Transparent particles that Anisotropy, if it occurs, introduces an arbitrary element are much larger than the wavelength of the illumination in scatter and fluorescence measurements. Calibration of behave like lenses, so that their scatter distribution can be detectors, instruments, reagents, and fluorescence stan- derived from simple rules of reflection and refraction. For dards has been a long-time (and somewhat elusive) goal of smaller particles, the effects of diffraction and the associ- the flow community. With anisotropy, two instruments ated constructive and destructive interference must also can yield different results, and both can be right. The be included. Consequently, the scatter from microscopic converse is also true. If machine parameters are adjusted objects may have very complicated, multi-lobed angular until two supposedly identical experiments yield the same distributions. Theoretical calculations can successfully results, artifacts such as offsets and nonlinearities may be predict these distributions for objects with simple shapes introduced. It is clear that we need to know how preva- and homogeneous refractive indices (18). However, bio- lent anisotropy of scatter and fluorescence is before flow logical cells are uniform and homogeneous by approxima- measurements can be truly standardized. tion only, often having rough surfaces and complex inter- In order to determine the extent to which cytometric nal structures. Furthermore, in flow cytometry, cells have measurements are affected by anisotropy, we need a the- an uncertain orientation and are observed in motion and ory and adequate measurement tools. This manuscript the illumination beam and scatter distribution are dis- presents simple theoretical models for scatter and fluores- torted by the fluid jet and other optical components. cence that predict how the polarization and emission Theoretical calculations incorporating all these effects anisotropy for these signals depend on experimental con- would be accurate but impractically cumbersome. Thus, ditions. A simple method to measure polarization is de- although the physical mechanisms of light scattering are scribed. It requires minimal modification of a flow cytom- well understood, theoretical predictions about scatter sig- eter. We then present measurements of calibration beads nals from individual cells in flow cytometry are approxi- to show that polarization in flow cytometry follows the mations that must be verified by experimentation. qualitative and quantitative predictions of the models. Theory, supported